Ankyrin (Ank) is an "adapter" that localizes protein complexes in functional sites within cells. There are three known Ank genes, Ank1 (erythroid), Ank2 (brain), and Ank3 (general) with overlapping tissue expression but cell type specific localization and binding preferences. Of the four domains--membrane binding, spectrin binding, death, and regulatory--the COOH-terminal regulatory region is the most divergent, provides the highest numbers of alternatively spliced transcripts, and is the least well characterized. There are four possible Ank1 COOH-termini that are conserved between mouse and man and show tissue and developmental specific expression. In the current proposal we will continue to determine the structure, localization, binding partners and function of the variant Ank1 isoforms. Isoform discovery is aided by two Ank1 mutations. 1) A spontaneous single base deletion in nb/nb Ank1 eliminates the regulatory region but leaves membrane and spectrin binding domains intact. The p150 nb-Ank1 generated modulates the severity of the Hereditary Spherocytosis by apparent binding to the membrane, but does not protect from loss of Purkinje cells in the cerebellum. Purkinje cell loss in nb/nb mice appears to be secondary to the onset of ataxia and alterations in the granule cell layer. 2) A knockout of the novel Ank1 encoded entirely within the regulatory region eliminates Ank1/17.5 but upregulates three other small isoforms. These are present in both Ank1/17.5-/- and nb/nb tissues indicating their origin from within the regulatory region. Ank1 in skeletal muscle binds to the sarcoplasmic reticulum. We hypothesize that Ank1 isoforms are structurally and functionally distinct and can modulate disease symptoms. We propose to: 1) Define the function of the alternate COOH-termini and the pl50 nb-Ank1 by add back experiments and functional assays; 2) Identify Ank1 containing structures in the granule cell and Purkinje cell layers by co-localization studies; 3) Define and characterize additional Ank1 isoforms by RT-PCR, 5'RACE, Real time PCR, and cDNA cloning; and 4) Determine the role of the ANK1/17.5 isoform in the structure and assembly of the sarcoplasmic reticulum by an in vivo developmental study.